Three cell lines, that is, the human breast cancer cell line (MCF-7) and the human mammary epithelial cell line (S-1) and its malignant form (T4-2) were embedded in a reconstituted basement membrane (Matrigal) that had 20-nL pyramid-shaped silicon microstructures. The proliferative behaviour of the MCF-7 cells was dependent on the surrounding conditions (2-D, collagen gel, or Matrigel), whereas the respiratory activity of a single cell (F-c) was almost identical under different culture conditions. The F-c value changed with cellular polarity. The F-c value for the S-1 cells was observed to decrease slightly, whereas that of the T4-2 cells increased 2 days after cultivation in the microstructures within the Matrigel. However, when the T4-2 cells were cultured in the presence of tyrphostin AG 1478 (T4-2 tyr) to inhibit epidermal growth factor (EGF) signaling, the F-c value decreased slightly and remained almost constant for an additional 1 week; this was similar to the behaviour of the S-1 cells. Further, fluorescence images showed that the T4-2 tyr cells formed polar structures that were similar to those formed by the S-1 cells whereas the T4-2 cells did not form such structures. These results indicate that cellular polarity can be assessed by measuring cellular respiratory activity.